Knockdown Macrophages Research Articles

Selenoprotein S maintains intestinal homeostasis in ulcerative colitis by inhibiting necroptosis of colonic epithelial cells through modulation of macrophage polarization.

Rationale: Macrophage polarization plays an important role in the inflammatory regulation of ulcerative colitis (UC). In this context, necroptosis is a type of cell death that regulates intestinal inflammation, and selenoprotein S (SelS) is a selenoprotein expressed in intestinal epithelial cells and macrophages that prevents intestinal inflammation. However, the underlying mechanisms of SelS in both cell types in regulating UC inflammatory responses remain unclear. Therefore, the direct effect of SelS deficiency on necroptosis in colonic epithelial cells (CECs) was investigated. In addition, whether SelS knockdown exacerbated intestinal inflammation by modulating macrophage polarization to promote necroptosis in CECs was assessed. Methods: The UC model of SelS knockdown mice was established with 3.5% sodium dextran sulfate, and clinical indicators and colon injury were evaluated in the mice. Moreover, SelS knockdown macrophages and CECs cultured alone/cocultured were treated with IL-1β. The M1/M2 polarization, NF-κB/NLRP3 signaling pathway, oxidative stress, necroptosis, inflammatory cytokine, and tight junction indicators were analyzed. In addition, co-immunoprecipitation, liquid chromatography-mass spectrometry, laser confocal analysis, and molecular docking were performed to identify the interacting proteins of SelS. The GEO database was used to assess the correlation of SelS and its target proteins with macrophage polarization. The intervention effect of four selenium supplements on UC was also explored. Results: Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) was identified as a potential interacting protein of SelS and SelS, Uba52, and yes-associated protein (YAP) was associated with macrophage polarization in the colon tissue of patients with UC. SelS deficiency in CECs directly induced reactive oxygen species (ROS) production, necroptosis, cytokine release, and tight junction disruption. SelS deficiency in macrophages inhibited YAP ubiquitination degradation by targeting Uba52, promoted M1 polarization, and activated the NF-κB/NLRP3 signaling pathway, thereby exacerbating ROS-triggered cascade damage in CECs. Finally, exogenous selenium supplementation could effectively alleviate colon injury in UC. Conclusion: SelS is required for maintaining intestinal homeostasis and that its deletion enhances necroptosis in CECs, which is further exacerbated by promoting M1 macrophage polarization, and triggers more severe barrier dysfunction and inflammatory responses in UC.

Abstract P3018: SCARNA20 Promotes M2 Macrophage Polarization Under Endotoxemia And Reduces Inflammation

Background: Macrophage polarization plays a key role in the inflammatory process. M1 pro-inflammatory and M2 anti-inflammatory phenotypes can down-regulate each other and may contribute to the pathogenesis of various diseases. Small Cajal Body Associated RNAs (SCARNAs) play a key role in alternative splicing and maturation of mRNAs. Our recent studies found that a combination of Procainamide (P, DNMT1 inhibitor) and Tubastatin A (T, HDAC6 inhibitor) reduces inflammation upon lipopolysaccharide (LPS) stimuli. Hypothesis: We hypothesize that the P+T treatment increases the expression of SCARNA20 hence reducing the proinflammatory response and promoting M2 macrophage polarization after LPS challenge. Methods and Results: To elucidate SCARNAs of interest during the inflammatory response, we treated macrophages with LPS (1μg/mL) and P (400nM/mL) + T (2.5μM/mL) for 1hr and performed RNA sequencing. Based on the sequencing results and qRT-PCR validation, SCARNA20 was the most significantly differentially expressed SCARNA between conditions (Fig. A) . The expression of TNFα was significantly reduced with the P+T treatment (Fig. B) . SCARNA20 overexpression (OE) showed an increase in the expression of the M2 marker CD163 and a reduction in the expression of the M1 marker CD80 (Fig. C) . Additionally, TNFα expression was significantly reduced in the SCARNA20 OE samples after the LPS challenge without treatment with P+T (Fig. D) . Finally, SCARNA20 knockdown macrophages (Fig. E) treated with P+T did not suppress TNFα expression (Fig. F) . Conclusions: We report for the first time that SCARNA20 is an important regulator of M2 macrophage polarization and pro-inflammatory response.

The prohibitin complex regulates macrophage fatty acid composition, plasma membrane packing, and lipid raft-mediated inflammatory signaling

Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure. SummaryProhibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.

Small extracellular vesicles from Ptpn1-deficient macrophages alleviate intestinal inflammation by reprogramming macrophage polarization via lactadherin enrichment

Tyrosine-protein phosphatase non-receptor type 1 (Ptpn1) is known to be involved in macrophage polarization. However, whether and how Ptpn1 regulates macrophage phenotype to affect intestinal epithelial barrier function remains largely unexplored. Herein, we investigated the impact of Ptpn1 and macrophage-derived small extracellular vesicles (sEVs) on macrophage-intestinal epithelial cell (IEC) interactions in the context of intestinal inflammation. We found that Ptpn1 knockdown shifts macrophages toward the anti-inflammatory M2 phenotype, thereby promoting intestinal barrier integrity and suppressing inflammatory response in the macrophage-IEC co-culture model. We further revealed that conditioned medium or sEVs isolated from Ptp1b knockdown macrophages are the primary factor driving the beneficial outcomes. Consistently, administration of the sEVs from Ptpn1-knockdown macrophages reduced disease severity and ameliorated intestinal inflammation in LPS-challenged mice. Furthermore, depletion of macrophages in mice abrogated the protective effect of Ptpn1-knockdown macrophage sEVs against Salmonella Typhimurium infection. Importantly, we found lactadherin to be highly enriched in the sEVs of Ptpn1-knockdown macrophages. Administration of recombinant lactadherin alleviated intestinal inflammation and barrier dysfunction by inducing macrophage M2 polarization. Interestingly, sEVs lactadherin was also internalized by macrophages and IECs, leading to macrophage M2 polarization and enhanced intestinal barrier integrity. Mechanistically, the anti-inflammatory and barrier-enhancing effect of lactadherin was achieved by reducing TNF-α and NF-κB activation. Thus, we demonstrated that sEVs from Ptpn1-knockdown macrophages mediate the communication between IECs and macrophages through enrichment of lactadherin. The outcome could potentially lead to the development of novel therapies for intestinal inflammatory disorders.

IMMU-35. TREM2 RESTRAINS ANTI-TUMOR CELL ACTIVITY OF MYELOID CELLS IN GLIOBLASTOMA

Abstract Tumor-associated myeloid cell populations occupy a major part of the glioblastoma (GBM) tumor microenvironment (TME). The prevailing view is that myeloid cells in the TME are immunosuppressive and promote GBM tumor progression. However, myeloid cells have the functional plasticity to either restrict or support tumor cell growth. TREM2 has been shown to alter the myeloid cell landscape in cancers arising in the body and plays important roles in brain microglial function in neurodegenerative diseases. But the role of TREM2 in the GBM TME and specifically in myeloid cell function has not been examined. Here we found that TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors, and that high TREM2 expression is associated with poor prognosis in GBM patients. TREM2 loss of function in human macrophages and mouse myeloid cells increased tumoricidal capacity in vitro using patient-derived glioblastoma stem cells and mouse glioblastoma cells. Accordingly, we found TREM2 in myeloid cells restricts proinflammatory polarization in both LPS-induced innate and IFNγ-induced adaptive immunity in vitro, mainly through the inhibition of NFκB and MAPK p38 signaling pathways. Orthotopic injection of mouse glioblastoma cells into TREM2 knockout mice increased animal survival compared to littermate wildtype mice. In addition, co-implantation of TREM2 knockdown macrophages or microglia with mouse glioblastoma cells in brains of immunocompetent mice increased animal survival compared to co-implantation of control myeloid cells or injection of tumor cells alone, suggesting an important role for TREM2 in myeloid cells in GBM growth in vivo. Together, these data indicate that TREM2 operates by restricting the anti-tumor and proinflammatory function of myeloid cells in GBM and that inhibition of the TREM2 pathway may represent a potential therapeutic strategy for these patients.

FBXO6 regulates the antiviral immune responses via mediating alveolar macrophages survival.

Inducing early apoptosis in alveolar macrophages is one of the strategies influenza A virus (IAV) evolved to subvert host immunity. Correspondingly, the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 is reported to interact with virus polymerase basic protein 1-frame 2 (PB1-F2) accessory protein to counteract virus-induced apoptosis. Herein, we report that one of the F-box proteins, FBXO6, promotes proteasomal degradation of NLRX1, and thus facilitates IAV-induced alveolar macrophages apoptosis and modulates both macrophage survival and type I interferon (IFN) signaling. We observed that FBXO6-deficient mice infected with IAV exhibited decreased pulmonary viral replication, alleviated inflammatory-associated pulmonary dysfunction, and less mortality. Analysis of the lungs of IAV-infected mice revealed markedly reduced leukocyte recruitment but enhanced production of type I IFN in Fbxo6-/- mice. Furthermore, increased type I IFN production and decreased viral replication were recapitulated in FBXO6 knockdown macrophages and associated with reduced apoptosis. Through gain- and loss-of-function studies, we found lung resident macrophages but not bone marrow-derived macrophages play a key role in the differences FBXO6 signaling pathway brings in the antiviral immune response. In further investigation, we identified that FBXO6 interacted with and promoted the proteasomal degradation of NLRX1. Together, our results demonstrate that FBXO6 negatively regulates immunity against IAV infection by enhancing the degradation of NLRX1 and thus impairs the survival of alveolar macrophages and antiviral immunity of the host.

KLK12 Regulates MMP-1 and MMP-9 via Bradykinin Receptors: Biomarkers for Differentiating Latent and Active Bovine Tuberculosis.

It has been established that kallikrein12 (KLK12) expression is closely related to bovine tuberculosis (bTB) development. Herein, we sought to clarify the regulatory mechanism of KLK12 and its application in tuberculosis diagnosis. KLK12 knockdown macrophages were produced by siRNA transfection. Bradykinin receptors (BR, including B1R and B2R) were blocked with specific inhibitors. Mannose-capped lipoarabinomannan (ManLAM) was extracted from Mycobacterium bovis (M. bovis) and used to study the mechanism of KLK12 activation. In addition, we constructed different mouse models representing the latent and active stages of M. bovis infection. Mouse models and clinical serum samples were used to assess the diagnostic value of biomarkers. Through the above methods, we confirmed that KLK12 regulates MMP-1 and MMP-9 via BR. KLK12 upregulation is mediated by the M. bovis-specific antigen ManLAM. KLK12, MMP-1, and MMP-9 harbor significant value as serological markers for differentiating between latent and active bTB, especially KLK12. In conclusion, we identified a novel signaling pathway, KLK12/BR/ERK/MMPs, in M. bovis-infected macrophages, which is activated by ManLAM. From this signaling pathway, KLK12 can be used as a serological marker to differentiate between latent and active bTB. Importantly, KLK12 also has enormous potential for the clinical diagnosis of human tuberculosis (TB).

N1-Methyladenosine (m1A) Regulation Associated With the Pathogenesis of Abdominal Aortic Aneurysm Through YTHDF3 Modulating Macrophage Polarization.

ObjectivesThis study aimed to identify key AAA-related m1A RNA methylation regulators and their association with immune infiltration in AAA. Furthermore, we aimed to explore the mechanism that m1A regulators modulate the functions of certain immune cells as well as the downstream target genes, participating in the progression of AAA.MethodsBased on the gene expression profiles of the GSE47472 and GSE98278 datasets, differential expression analysis focusing on m1A regulators was performed on the combined dataset to identify differentially expressed m1A regulatory genes (DEMRGs). Additionally, CIBERSORT tool was utilized in the analysis of the immune infiltration landscape and its correlation with DEMRGs. Moreover, we validated the expression levels of DEMRGs in human AAA tissues by real-time quantitative PCR (RT-qPCR). Immunofluorescence (IF) staining was also applied in the validation of cellular localization of YTHDF3 in AAA tissues. Furthermore, we established LPS/IFN-γ induced M1 macrophages and ythdf3 knockdown macrophages in vitro, to explore the relationship between YTHDF3 and macrophage polarization. At last, RNA immunoprecipitation-sequencing (RIP-Seq) combined with PPI network analysis was used to predict the target genes of YTHDF3 in AAA progression.ResultsEight DEMRGs were identified in our study, including YTHDC1, YTHDF1-3, RRP8, TRMT61A as up-regulated genes and FTO, ALKBH1 as down-regulated genes. The immune infiltration analysis showed these DEMRGs were positively correlated with activated mast cells, plasma cells and M1 macrophages in AAA. RT-qPCR analysis also verified the up-regulated expression levels of YTHDC1, YTHDF1, and YTHDF3 in human AAA tissues. Besides, IF staining result in AAA adventitia indicated the localization of YTHDF3 in macrophages. Moreover, our in-vitro experiments found that the knockdown of ythdf3 in M0 macrophages inhibits macrophage M1 polarization but promotes macrophage M2 polarization. Eventually, 30 key AAA-related target genes of YTHDF3 were predicted, including CD44, mTOR, ITGB1, STAT3, etc.ConclusionOur study reveals that m1A regulation is significantly associated with the pathogenesis of human AAA. The m1A “reader,” YTHDF3, may participate in the modulating of macrophage polarization that promotes aortic inflammation, and influence AAA progression by regulating the expression of its target genes.

GRIM19 downregulation-induced pyroptosis of macrophages through NLRP3 pathway in adenomyosis

Research questionDoes the absence of GRIM19 affect pyroptosis of macrophages? Is the release of IL-1β caused by pyroptosis a relevant factor in the regulation of adenomyosis progression? DesignEndometrial tissues were collected from patients with (n = 12) and without (n = 12) adenomyosis. GRIM19 expression of adenomyosis tissues was analysed by western blot and real-time polymerase chain reaction (RT-PCR). In GRIM19 knockdown macrophages, pyroptosis-related factors expressions were also measured by western blot and RT-PCR. The human endometrial stromal cells (HESC) were co-cultured with GRIM19-depleted macrophages and IL-1β neutralizing antibody to detect the effects of pyroptosis of macrophages on apoptosis, proliferation and migration of HESC. ResultsThe expression of GRIM19 was significantly lower in adenomyosis (P = 0.0002). In THP-1-derived macrophages, the expression of NLRP3 (P < 0.0001), ASC (P = 0.0176), caspase-1 (P = 0.0368), GSDMD (P = 0.0453) and IL-1β (P = 0.0208) are increased after downregulation of GRIM19. GRIM19 knockdown induced the release of IL-1β (P = 0.0195) in THP-1-derived macrophages. The apoptosis of HESC co-cultured with GRIM19 knockdown macrophages was significantly inhibited (P < 0.0001), the proliferation (P = 0.0254) and migration (P < 0.0001) were markedly promoted. Existence of IL-1β neutralizing antibody in supernatants recovered the effects (P < 0.0001) of GRIM19 knockdown macrophages on HESC. ConclusionsGRIM19 downregulation induces pyroptosis of macrophages through NLRP3 pathway, increases the secretion of IL-1β and promotes adenomyosis progression.

The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function.

Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection.

Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation.

Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability to genetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.

Global Transcriptomics Uncovers Distinct Contributions From Splicing Regulatory Proteins to the Macrophage Innate Immune Response

Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

The protective role of YTHDF1-knock down macrophages on the immune paralysis of severe sepsis rats with ECMO

ObjectiveTo examine the role of YTHDF1 knock-down macrophages on the immunity of severe sepsis rats with ECMO. Methods15 SD rats were randomly allocated into 3 groups: mild sepsis (I), severe sepsis with ECMO (II), and YTHDF1 knock-down macrophages treatment groups (III). Blood biochemical indexes, different immune factors and brain changes were detected by RT-PCR, ELISA, ELISPOT and HE staining. Isolated macrophages subtypes and signal proteins were detected by flow cytometry, western blot and m6A RNA methylation test. ResultsThe levels of HMGB1, RAGE, YTHDF1 and IL-17 in peripheral blood were significantly higher (p < 0.01), while the level of CXCL9 and TNF-α, and LPS-specific CD8+CTL function were significantly decreased in group II compared with group I (p < 0.01). The ratio of CD63+ macrophages (p < 0.05) and CD64+ macrophages (p< 0.05) decreased and the level of elastase (p < 0.01) and CCR2highCX3CR1low/CCR2lowCX3CR1high (p < 0.01) of macrophages increased in group II. The above were consistent with the severity of biochemical indicators, the increasing endothelial injury factor (Ang2/Ang1), lower endothelial protective factor (sTie2), severer brain injury in group II. After YTHDF1 knock-down macrophages treatment, the above indexes' changes were opposite when Group III versus Group II through the down-regulation of m6A RNA methylation of JAK2/STAT3 (p < 0.01) and protein expression of PJAK2/PSTAT3 (p < 0.05) in isolated macrophages. ConclusionsYTHDF1 knock-down macrophages improved the immune paralysis of macrophages, Th1/Th17 and CTL and reduced the entry of macrophages into the brain to cause endothelial damage of severe sepsis rats with ECMO through the inhibition of HMGB1/RAGE and YTHDF1, m6A RNA methylation of JAK2/STAT3 and PJAK2/PSTAT3 proteins expression in macrophages.

Alternative splicing of Bax controls cell death and innate immune responses in macrophages

Abstract To defend against invading pathogens, the macrophage innate immune response requires rapid, robust gene expression reprogramming. Innate immune genes that are induced as part of this response are regulated at many steps. While transcriptional activation of innate immune genes has been extensively studied, the mechanisms driving their post-transcriptional regulation (i.e. alternative splicing) needs further investigation. To begin learning how pre-mRNA splicing changes impact the innate immune response in macrophages, we examined how loss of a splicing regulatory protein called SRSF6 (serine arginine rich splicing factor 6) impacts gene expression. We discovered a novel phenotype whereby loss of SRSF6 increased basal expression of many interferon stimulated genes (ISGs). Consistent with mitochondrial dysfunction, a phenotype that we and others have previously associated high basal ISG expression, we measured an increase in apoptosis and macrophage depolarization in SRSF6 knockdown macrophages compared to scramble controls. Subsequent bioinformatic analysis of RNA-seq data from SRSF6 knockdown macrophages identified an alternative splicing event in the apoptosis activation factor BAX. While Bax isoforms have previously been linked to cell death regulation, no one has reported a role for Bax isoforms in modulating mtDNA-driven innate immune responses. Our data is consistent with a model whereby upregulation of a Bax isoform promotes pore formation in mitochondria which 1) causes leakage of mtDNA into the cytosol where it is detected by cGAS, eliciting type I interferon production and 2) primes macrophages to undergo programmed cell death.

Activating transcription factor 3 involved in Pseudomonas aeruginosa PAO1-induced macrophage senescence

Pseudomonas aeruginosa (PA) is one of the most prevalent pathogens that cause nosocomial infection in critical patients. Previously, we reported PA induced macrophage to senescence under the circumstance of infection. As an oxidative stress responsiveness element, activating transcription factor 3 (ATF3) might be involved in the macrophage senescence process. To test this presumption, we manipulated the expression of ATF3 in macrophage by using a PAO1 infection system. In the present study, ATF3 expression in macrophage was increased, following the duration and colony counts of PAO1 infection. Knockdown of ATF3 in macrophage resulted in increased percentage of senescent macrophage under PAO1 infection, while overexpressing ATF3 partly blocked PAO1-induced macrophage senescence. In accordance with the senescent phenotype, elevated reactive oxygen species (ROS) production was shown in ATF3 knockdown macrophages. Also, capacity of phagocytosis was also affected by manipulation of ATF3 expression in macrophages, and increased phagocytosed fluorescent beads was found in ATF3 knockdown macrophage. ATF3 might regulate the senescence process through influence on NF-κB translocation. During infection, the overexpression or downregulation of ATF3 in macrophage negatively modulated the translocation of NF-κB p65 and its phosphorylation at Ser-536. As a result, IL-6 and TNFα was elevated, while IL-10 decreased in case of ATF3 knockdown. In conclusion, ATF3 negatively regulates NF-κB translocation and activation, and participates in PA-induced macrophage senescence. As oxidative stress and inflammation induced element, ATF3 may modulate macrophage-related host defense.

Kallikrein 12 regulates innate resistance of murine macrophages against Mycobacterium bovis infection by modulating autophagy and apoptosis

Aims and objectives: Tuberculosis (TB) is a leading infectious cause of mortality worldwide and millions of people continue to develop active TB each year. Mycobacterium bovis (M. bovis) is a member of Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis and imposing high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases and their role is established in various physiological and pathological processes. Since the KLKs expression has been linked to various cancerous, degenerative and infectious conditions, it is likely that KLKs expression may mediate host immune response against M. bovis infection. Methods: In the current study, we investigated the expression of KLK12 in M. bovis infection in vivo and in vitro by using C57BL/6 mice and two types of murine macrophages, RAW264.7 cells and bone marrow derived macrophages (BMDMs). In this study, we used two strains of M. bovis, M. bovis C68004 strain and M. bovis N strain. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown BMDMs by using siRNA transfection. We investigated the expression of various proteins through QRT-PCR, Immunoblot and ELISA. Apoptotic cells were detected by flow cytometry. Furthermore, cell viability and phagocytic ability of macrophages was also determined after transfection and infection. CFU assay was performed to know the intracellular survival of M. bovis. Results: Our results exhibited an increase in the expression of KLK12 in the lung and spleen of M. bovis infected mice. A dose (MOI) and time dependent up-regulation was also noticed in RAW264.7 and BMDMs. Then, we evaluated the role of KLK12 in innate immune response regulation against M. bovis. Interestingly, knockdown of KLK12 resulted into significant down regulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1β, IL-6, IL-12 and TNF-α were also significantly down regulated in KLK12 knockdown macrophages but difference in IL-10 and IFN-β expression was non-significant. Our finding also depicted that knockdown of KLK12 enhances the intracellular survival of M. bovis. Conclusion: Taken together, these findings suggest that up-regulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine and human TB.

Pioglitazone-Mediated Attenuation of Experimental Colitis Relies on Cleaving of Annexin A1 Released by Macrophages.

Ulcerative colitis and Crohn’s disease are chronic inflammatory bowel diseases (IBDs) which burden health systems worldwide; available pharmacological therapies are limited and cost-intensive. Use of peroxisome proliferator activated-receptor γ (PPARγ) ligands for IBD treatment, while promising, lacks solid evidences to ensure its efficacy. Annexin A1 (AnxA1), a glucocorticoid-modulated anti-inflammatory protein, plays a key role on IBD control and is a potential biomarker of IBD progression. We here investigated whether effects of pioglitazone, a PPARγ ligand, rely on AnxA1 actions to modulate IBD inflammation. Experimental colitis was evoked by 2% dextran sodium sulfate (DSS) in AnxA1 knockout (AnxA1−/−) or wild type (WT) C57BL/6 mice. Clinical and histological parameters were more severe for AnxA−/− than WT mice, and 10 mg/kg pioglitazone treatment attenuated disease parameters in WT mice only. AnxA1 expression was increased in tissue sections of diseased WT mice, correlating positively with presence of CD68+ macrophages. Metalloproteinase-9 (MMP-9) and inactive 33 kDa AnxA1 levels were increased in the colon of diseased WT mice, which were reduced by pioglitazone treatment. Cytokine secretion, reactive oxygen species generation and MMP-9 expression caused by lipopolysaccharide (LPS) treatment in AnxA1-expressing RAW 264.7 macrophages were reduced by pioglitazone treatment, effects not detected in AnxA1 knockdown macrophages. LPS-mediated increase of AnxA1 cleaving in RAW 264.7 macrophages was also attenuated by pioglitazone treatment. Finally, pioglitazone treatment increased extracellular signal-regulated kinase (ERK) phosphorylation in AnxA1-expressing RAW 264.7 macrophages, but not in AnxA1-knockdown macrophages. Thus, our data highlight AnxA1 as a crucial factor for the therapeutic actions of pioglitazone on IBDs.

Epigenetic Studies Revealed a Ghost Proteome in PC1/3 KD Macrophages under Antitumoral Resistance Induced by IL-10.

Our previous investigation on macrophages has allowed us to show that the inhibition of the enzyme proprotein convertase (PC1/3) controls the activation of macrophages. We demonstrated that PC1/3 knockdown (KD) in macrophages exhibits an increased secretion of proinflammatory and antitumoral factors. In this biological context, we assessed the presence of histone modifications and the presence and contribution of a “ghost proteome” in these macrophages. We identified a set of alternative proteins (AltProts) that have a key role in the regulation of various signaling pathways. In this study, to further investigate the underlying mechanisms involved in the resistance of PC1/3-KD macrophages to anti-inflammatory stimuli, we have conducted a proteomic system biology study to assess the epigenome variation, focusing on histone modifications. Results from our study have indicated the presence of significant variations in histone modifications along with the identification of 28 AltProts, which can be correlated with antitumoral resistance under IL-10 stimulation. These findings highlight a key role of altered epigenome histone modifications in driving resistance and indicate that like the reference proteins, AltProts can have a major impact in the field of epigenetics and regulation of gene expression, as shown in our results.

Recombinant Ricin Toxin Binding Subunit B (RTB) Stimulates Production of TNF-α by Mouse Macrophages Through Activation of TLR4 Signaling Pathway.

Ricin toxin binding subunit B (RTB) is a galactose-binding lectin protein derived from the beans of the castor oil plant (Ricinus communis). Our previous studies have reported a direct immunomodulatory effect of recombinant RTB, which stimulates RAW264.7 cells to produce cytokines including TNF-α. However, the role of RTB in innate immune response and its specific mechanism have not been reported in detail. In this work, the results showed that RTB treatment of macrophages significantly increased TLR4 protein levels. RTB also activated TLR4 downstream events, including MyD88, IRAK, and TRAF6, resulting in macrophage activation and TNF-α production. This process is reflected in the increase of IκB phosphorylation. TLR4 knockdown macrophages treated with RTB exhibited greatly reduced IκB phosphorylation and TNF-α secretion. Moreover, treatment with MyD88 inhibitor also suppressed TNF-α production. The docking of RT and TLR4 was simulated by computer, and the contact residues were concentrated on RTB. Our results suggest that recombinant RTB can activate mouse macrophages to secrete TNF-α through activation of NF-κB via the TLR4 signaling pathways.

Sestrin1 inhibits oxidized low-density lipoprotein-induced activation of NLRP3 inflammasome in macrophages in a murine atherosclerosis model.

Macrophages play a crucial role in the progression of atherosclerotic lesions. In the current study, we analyzed the expression and function of sestrin1 (SESN1) in the aorta macrophages in a murine atherosclerosis model. We identified high SESN1 expression in the aorta macrophages in atherosclerotic mice. Using lentivirus-mediated SESN1 overexpression in macrophages, we found that SESN1 inhibited oxidized low-density lipoprotein-induced NLRP3 inflammasome activation in lipopolysaccharide (LPS)-primed macrophages, as evidenced by less ASC-NLRP3 complex formation, lower caspase-1 activation, and lower generation of mature IL-1β. Besides, SESN1 impeded oxidized low-density lipoprotein-induced activation of NK-κB signaling in macrophages. Furthermore, SESN1 suppressed cholesterol crystal-induced NLRP3 inflammasome activation and foam cell formation. Adoptive transfer of SESN1 overexpressing macrophages reduced the expression of pro-inflammatory cytokines in infiltrating macrophages and the whole aorta tissue. Adoptive transfer of SESN1 knockdown macrophages enhanced the expression of pro-inflammatory cytokines in infiltrating macrophages and the whole aorta tissue. Overall, our study sheds light on the significance of SESN1 for macrophage-mediated aorta inflammation.